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Abstract

In two long term studies of non-Hodgkin's Lymphoma (NHL), the only clinical feature associated with a high relapse rate and treatment resistance is the presence of the B-cell lymphoma/leukemia-2(bcl-2) proto-oncogene in an over-active state. Regulation of this gene has shown promise as a means for better treatment in patients with relapsed NHL. At MU, in the past, many antisense and nonsense peptide-peptide nucleic acid (PNA) conjugates that target the bcl-2 proto-oncogene were synthesized and radiolabeled for mRNA binding evaluations.
This summer, we synthesized two new peptide-peptide nucleic acid conjugates. The first of these was a nonsense sequence of PNA monomers attached to the peptide Tyrosine-3-Octreotate (1), and the second was the anti-bcl-2 sequence attached to Alanine-box Octreotate (2). The PNA sequences attached to the peptides correspond to the first fourteen bases of the bcl-2 proto-oncogene (ccagcgtgcgccat) in the case of the anti-bcl-2 compound (2) and correspond to no matching sequence in the human genome in the case of the nonsense sequence (1). They are both for later use as a negative control in mRNA binding evaluations (the previously synthesized positive agent being anti-bcl-2 attached to Tyrosine-3-Octreotate). The peptides were synthesized using standard Fmoc solid phase peptide synthesis on a resin using a very low substitution level. The peptides were synthesized in an automatic peptide synthesizer, and then elongated with the addition of the PNA monomers manually in a reaction vessel. Each peptide-PNA construct was then coupled to DOTA (1, 4, 7, 10-tetraazacyclododecane-N, N', N'', N'''-tetraacetic acid), a ligand to provide a site for subsequent radiometal chelation. The correct molecular weight with a high purity was observed in the LC-MS results for the peptide-PNA compound 1 after two attempts, and compound 2 was successful after the first attempt. The amount of each construct synthesized should be enough for the later mRNA binding study.